CONICET | Buscador de Institutos y Recursos Humanos

Aquaporins are tetrameric channels that facilitate the osmotic diffusion of waterand small molecules across membranes. Plasma membrane intrinsic proteins (PIP)represent the larger subfamily in higher plants, and is divided into two paralogues,PIP1 and PIP2, able to form both homo and heterotetramers (Fetter et al., PlantCell, 2004). Previous results show that BvPIP are pH sensitive channels, with PIP2-PIP1 heterotetramers pH0,5 shifted to acidic values in comparison with PIP2homotetramer sensing (Yaneff et al., PNAS 2014; Jozefkowicz et al., Biophys J2016). A mechanistic gating model was proposed for these channels on the basisof SoPIP2;1 X-ray crystallography. This mechanism involves conformationalchanges in loop D, N- and C-terminal domains. However, even if this modelproposes C-terminal domain as a key element of the gating mechanism, the lastpart of this flexible domain could not be assigned in the crystal structure due to itsdisorder. As the length of C-terminal domain is one of the characteristic differencesbetween PIP1 and PIP2, we investigated if this domain is relevant for PIP gatingmechanism.We generated two PIP2 mutations : 1) the deletion of the last six C-terminalresidues, mimicking the length of PIP1, and 2) the replacement of the C-terminaldomain with the corresponding fragment of PIP1. We assayed the biologicalactivity and pH regulation of PIP2 mutants expressed alone or co-expressed withPIP1. Interestingly, we found that mutations at the C-terminal domain canmarkedly modify PIP2 pH gating. Our results suggest a capital role of this domainin the pH gating mechanism that couldn´t been resolved with structural data.These new findings shed light on the C-terminal relevance on pH gating and openthe possibility of a deeper understanding of PIP gating mechanism.